Spectral karyotyping is an evolving cytogenetic technology to identify chromosomal rearrangements such as deletions, inversions, and translocations seen in cancer cell lines. A commercial instrument operates as a modified Sagnac interferometer installed on an epi- illumination microscope to capture the spectral signature of probes used to paint chromosomes. The epi-illumination port and the fluorescent cube assembly of the microscope have been modified to permit single wavelength excitation, resulting in greater than an order of magnitude improvement in the signal to noise ratio of the fluorescent signals and a reduction in the overall background signal. These improvements will permit resolution of smaller chromosome band abnormalities and identification of their correlation with disease. - SKY, spectral karyotyping, cytogenetics, chromosomal abnormality, instrumentation